A multidasciplinary study aimed at uncovering the structural and energetic basis for amino acid and transfer RNA specificity in several class I aminoacyl-tRNA synthetases is proposed. The experimental plan will focus on the structure-based design of directed modifications in both the synthetase and tRNA, and on the insightful analysis of modified complexes by a variety of enzymological and biophysical methods. The role of the globular tertiary core region of glutamine-specific tRNA in aminoacylation will be explored by exploiting the availability of an extensive tRNA sequence database, as well as by the introduction of highly specific chemical modifications to the sugar-phosphate backbone. Glutaminylation kinetics, together with high-resolution structures bound to synthetase and to Ef-Tu, will be determined to assess the effects of the alterations. In a separate line of experiments, rational structure-based approaches are planned to re-engineer the amino acid specificity from glutamine to glutamate. New methodologies will be applied to improve the chemical and enzymatic synthesis of multimilligram quantities of homogeneous RNAs, and to better characterize the aminoacylation reaction by implementing a new assay useful for both steady-state and transient kinetic measurements. With improved kinetic methods in hand, a functional analysis of glutaminyl-tRNA synthetase will be pursued with the aim of addressing the kinetic basis for the amino acid specificity, the function of divalent metal ions, and the requirement for tRNA to carry out activation of the amino acid. These approaches will also be used to examine kinetic properties of the homologous cysteinyl-tRNA synthetase. Detailed information on the origins of class I bacterial tRNA synthetase specificities will aid drug-design approaches which exploit the structural differences between bacterial and human synthetases to develop antimicrobial agents. Re-engineering of the amino acid specificities of tRNA synthetases is also the most challenging step in the in viva production of proteins containing non-natural amino acids. Because of the broad applicability of this technology, the development of new synthetases based on the class I active site domain has potential for impact on biomedical research toward therapies for many human diseases.